US Department of Health and Human Services. Reducing the Health Consequences of Smoking: A Report of the
Surgeon General . Rockville, Md: US Dept of Health and Human Services, Public Health
Service, Centers for Disease Control, Center for Chronic Disease Prevention
and Health Promotion, Office on Smoking and Health; 1989.

Desenclos JCA, Hahn RA.Centers for Disease Control and Prevention. Years of potential life lost before age 65, by race Hispanic origin,
and sex—United States, 1986-1988. Mor Mortal Wkly Rep CDC Surveill Summ.1992;41(SS-6):13-23.

Nicotine Metabolism and Intake in Black and White Smokers

From the Division of General Internal Medicine, Department of Medicine, Medical Effectiveness Research Center for Diverse Populations (Dr Pérez-Stable and Ms Herrera); Division of Clinical Pharmacology and Experimental Therapeutics, Medical Service, San Francisco General Hospital Medical Center (Drs Jacob and Benowitz); and Departments of Medicine and Psychiatry (Drs Jacob and Benowitz), University of California, San Francisco.

Objective.— To compare the rates of metabolism and the daily intake of nicotine
in black smokers and white smokers.

Design.— Participants received simultaneous infusions of deuterium-labeled nicotine
and cotinine. Urine was collected for determination of total clearance of
nicotine and cotinine, fractional conversion of nicotine to cotinine, and
cotinine elimination rate. Using cotinine levels during ad libitum smoking
and clearance data, the daily intake of nicotine from smoking was estimated.

Setting.— Metabolic ward of a university-affiliated public hospital.

Participants.— A total of 40 black and 39 white smokers, average consumption of 14
and 14.7 cigarettes per day, respectively, of similar age (mean, 32.5 and
32.3 years, respectively) and body weight (mean, 73.3 and 68.8 kg, respectively).

Main Outcome Measures.— Clearance (renal and nonrenal), half-life, and volume of distribution
of nicotine and cotinine and the calculated daily intake of nicotine.

Results.— The total and nonrenal clearances of nicotine were not significantly
different, respectively, in blacks (17.7 and 17.2 mL·min−1·kg−1) compared with whites (19.6 and 18.9 mL·min−1·kg−1) (P=.11
and .20). However, the total and nonrenal clearances of cotinine were significantly
lower, respectively, in blacks (0.56 and 0.47 mL·min−1·kg−1) than in whites (0.68 vs 0.61 mL·min−1·kg−1; P =.009 for each comparison). The
nicotine intake per cigarette was 30% greater in blacks compared with whites
(1.41 vs 1.09 mg per cigarette, respectively; P =.02).
Volume of distribution did not differ for the 2 groups, but cotinine half-life
was higher in blacks than in whites (1064 vs 950 minutes, respectively; P =.07).

Conclusions.— Higher levels of cotinine per cigarette smoked by blacks compared with
whites can be explained by both slower clearance of cotinine and higher intake
of nicotine per cigarette in blacks. Greater nicotine and therefore greater
tobacco smoke intake per cigarette could, in part, explain some of the ethnic
differences in smoking-related disease risks.

LUNG CANCER and chronic obstructive pulmonary disease (COPD) are primarily
diseases of cigarette smokers, with estimated smoking-attributable mortalities
in excess of 80% among both men and women.1
The incidence and mortality of lung cancer and COPD differ by race and sex
between blacks and whites.2- 4
Black men have a higher incidence of and mortality from lung cancer than do
white men, while rates among women are similar in these 2 racial groups.2,4 Differences in smoking rates and socioeconomic
status between blacks and whites explain most of the observed differences
in lung cancer incidence and mortality among men.2
However, available evidence indicates that blacks have a lower risk of developing
and dying from COPD compared with whites, and this discrepancy in racial differences
in smoking-related diseases is unexplained.2
In addition, the risk for low-birth-weight infants among black women is greater
than that for white women after adjusting for cigarette consumption.5,6

Cigarette-smoking behavior differs substantially by race and sex in
the United States.7 Blacks have had an overall
higher prevalence of cigarette smoking since 1965,7
but data for 1994 show similar overall rates, with 26.3% of whites and 27.2%
of blacks reporting current cigarette smoking.8
However, sex differences persist as black men continue to smoke at higher
rates than white men (33.9% vs 28.0%), while black women smoke at lower rates
than white women (21.8% vs 24.7%).8 Black smokers
smoke differently than whites with consistently fewer reported cigarettes
smoked per day compared with their white counterparts and a predominant preference
for mentholated cigarettes.7,9
Despite smoking fewer cigarettes per day, black smokers have higher levels
of serum cotinine, the proximate metabolite of nicotine, after controlling
for number and yield of cigarettes.10 This
observation raises the questions of racial differences in accuracy of self-reported
number of cigarettes smoked,11 in the intensity
of smoking cigarettes, and whether nicotine and/or cotinine are metabolized
differently in blacks compared with whites.

Racial differences in nicotine metabolism could be important in understanding
differences in smoking behavior and rates of some smoking-related diseases.
Cigarette smokers tend to regulate their tobacco consumption to gain the desired
effects of nicotine, which are related to the levels of nicotine in the body.12,13 Nicotine is extensively metabolized
in the liver, and there is considerable individual variability in the rate
of nicotine metabolism.14- 17
Persons who metabolize nicotine more rapidly would need to smoke more to maintain
the same level of nicotine in the body than those who are slower metabolizers
of nicotine.

The objective of our study was to compare the rates of metabolism of
nicotine and its metabolite cotinine in black smokers and white smokers. Based
on the metabolism data, we were also able to estimate the daily intake of
nicotine from cigarette smoking, which presumably reflects the intake of other
tobacco smoke toxins, in the same individuals.

METHODS

Participants

Volunteers were recruited through posted advertisements in the San Francisco,
Calif, black community and at local community colleges. Eligibility criteria
included (1) being in good health on the basis of history, physical examination,
electrocardiogram, and blood chemistries; (2) age 21 to 64 years; (3) male
or nonpregnant female defined by surgical sterilization or negative pregnancy
test result; and (4) self-identified as non-Latino white or black. Exclusion
criteria were habitual use of any prescription medication, narcotic or sedative
drug addiction, or long-term alcoholism. Eligible subjects received $250 for
completing the study successfully. Black subjects and white subjects were
matched by sex and age within 5 years. Subjects were also stratified by self-reported
cigarette consumption of 1 to 9 or 10 or more cigarettes per day.

Screening Procedure. Potentially eligible subjects completed a questionnaire on demographic
items, smoking history, current smoking behavior, medical history, and habitual
use of prescribed medications, alcohol, and other drugs. At the time of the
eligibility evaluation, blood was collected and analyzed for serum cotinine
as a measure of ad libitum nicotine intake from cigarette smoking and for
routine screening tests (complete blood cell count, chemical analyses of the
blood, evaluation for hepatitis B antigen) to determine eligibility. The screening
cotinine blood samples were collected from all participants in the afternoon.
Results of the questionnaires and blood tests were reviewed by a physician
prior to contacting the potential participant.

Experimental Procedure. Eligible subjects were asked to come to the clinical study center at
San Francisco General Hospital between 7 and 8 AM, at which time they completed
questionnaires on smoking behavior, including a scale to measure physical
dependence on tobacco.18 Subjects were asked
to abstain from food and cigarette smoking from 10 PM the previous night until
arrival at the clinical study center. Overnight abstinence from tobacco was
assessed by measurement of plasma concentration of nicotine (unlabeled) prior
to the infusion.

Venous catheters were placed in both forearms. Subjects received a simultaneous
infusion of deuterium-labeled nicotine-d2 (3′,3′-dideuteronicotine)
and cotinine-d4 (2,4,5,6-tetradeuterocotinine) for 30 minutes.
Smokers of 10 or more cigarettes per day received 2.0 µg·kg−1·min−1 of nicotine-d2 and
cotinine-d4; infusions of this dose result in nicotine blood levels
similar to those observed with cigarette smoking and are tolerated well by
habitual smokers.13,14 A modified
dose of 1.5 µg·kg−1·min−1 was administered to self-reported smokers of 1 to 9 cigarettes per
day. During all infusions, subjects were monitored by continuous electrocardiography
and frequent blood pressure measurements taken by an automated blood pressure
machine. Two hours after the end of the infusion, subjects were given a light
breakfast. Subjects were allowed to smoke their cigarettes freely after 1
PM or about 5 hours after the beginning of the labeled nicotine and cotinine
infusion.

Blood samples (5 mL) for measurement of nicotine and cotinine levels
were collected at 0, 10, 20, 30, 45, 60, 90, 120, 240, 360, and 480 minutes,
and then 24, 48, 72, and 96 hours after the infusion to include at least 3
half-lives for cotinine.13 Urine was collected
during the infusion and up to 480 minutes thereafter. The study had approval
of the University of California, San Francisco (UCSF), Committee on Human
Research.

Analysis of Nicotine and Cotinine in Biological Fluids. Analysis of blood samples for concentration of nicotine and cotinine
at the eligibility evaluation was performed by gas chromatography (GC) with
nitrogen-phosphorus detection.19 Assays of
samples collected during and after infusion of labeled nicotine and cotinine
were performed by GC with mass-selective detection.20
Gas chromatography–mass spectroscopy (GC-MS) was required because the
metabolic studies were performed using deuterium-labeled nicotine and cotinine.21 Labeled compounds are necessary for metabolic studies
because smokers already have considerable levels of nicotine and cotinine
in their bodies that would make measurements of clearance of unlabeled nicotine
or cotinine impossible. Internal and external quality-control procedures are
used routinely in the laboratory. Samples were frozen and assayed in batches.
The GC-MS assay sensitivity is 0.003 µmol/L (0.5 ng/mL) for nicotine
and 28 nmol/L (5 ng/mL) for cotinine. The GC assay previously described19 was modified for use with a capillary column and
simultaneous extractions and chromatography of nicotine and cotinine.20 The GC assay sensitivity for nicotine is 0.006 µmol/L
(1.0 ng/mL) and for cotinine is 57 nmol/L (10 ng/mL).

Nicotine and Cotinine Salts. Nicotine-d2 tartrate and cotinine-d4 base were
synthesized as described previously and purified by multiple recrystallizations
and purity certified by microanalysis (C,H,N), thin-layer chromatography,
and GC-MS.22 Deuterium-labeled nicotine and
cotinine were made up in 0.9% sodium chloride, sterilized by autoclaving,
and sealed under nitrogen in vials by the Pharmaceutical Preparations Laboratory
at UCSF. Specimens were pyrogen tested, and concentrations of nicotine and
cotinine were measured by GC prior to use in subjects.

Pharmacokinetic Analysis. Pharmacokinetic parameters were estimated from blood concentration and
urinary excretion data using model-independent methods described previously.23 The terminal elimination rate constant and half-life
were determined from the slope of the terminal log blood concentration–time
curve using linear least squares regression. Total clearances were computed
as CLnic=Dose(nic−d2)/AUC(nic−d2)
and CLcot=Dose(cot−d4)/AUC(cot−d4),
where CL is clearance; AUC, area under the curve; nic, nicotine; and cot,
cotinine. Renal clearances were calculated as urinary excretion of nicotine
or cotinine divided by the AUC, on urine collected for the 8 hours during
and after the infusion while subjects were on the research ward. Metabolic
clearance was estimated as total clearance minus renal clearance.

Fractional conversion of nicotine to cotinine (f) was estimated using
blood levels of cotinine generated from infused nicotine and the clearance
of cotinine itself, determined by infusion of cotinine: 22cot

Daily intake of nicotine from tobacco was estimated based on knowledge
of fractional conversion of nicotine to cotinine and total clearance of cotinine,
as described and validated previously.23 At
steady state, the estimation was based on the following: COTgen=COTelim=fDnic, where COTgen is the amount of cotinine
generated from nicotine; COTelim, the amount eliminated from the
body per day; f, the fractional conversion of nicotine to cotinine; and Dnic, the daily intake of nicotine. Cotinine elimination rate can be
estimated as the product of average plasma cotinine concentration (Ccot) during habitual cigarette smoking and total body clearance of cotinine
(CLcot): COTelim=Ccot CLcot. Combining
these 2 equations, Dnic=Ccot CLcot/f. Based
on each of these parameters, we estimated the daily intake of nicotine and,
using the reported daily cigarette consumption, nicotine intake per cigarette
for each subject.

Data Analysis. Means and SDs were calculated where appropriate and comparisons between
blacks and whites were performed using the t test
for continuous variables and χ2 test for categorical variables.24 Pharmacokinetic parameters were compared by a 2×2
analysis of variance, examining effects of race and sex.

RESULTS

A total of 79 subjects were studied. Demographic variables and smoking
behavior are shown in Table 1.
Mean age, the percentage of male participants, and average body weight were
similar for blacks and whites. All participants had normal blood pressure,
serum glucose and creatinine levels, and creatinine clearance. Blacks had
fewer years of education (12.8 vs 13.9 years; P =.08)
and were less likely to be employed (45% vs 64%; P=.02).
Although the number of cigarettes smoked per day, years of smoking, and Fagerstrom
Tolerance scores were similar by race, blacks on average reported a shorter
time to first cigarette after waking up and smoked cigarettes with a higher
content of nicotine, tar, and carbon monoxide as determined by machine testing
using the Federal Trade Commission method. Most blacks reported smoking mentholated
cigarettes, compared with only 2 whites.

Plasma concentrations of labeled nicotine and cotinine during and after
intravenous infusion were similar to previous studies with smokers.23 Pharmacokinetic parameters for nicotine among black
and white smokers are shown in Table 2.
The total and nonrenal clearance of nicotine was, on average, lower in blacks
than in whites, but these differences were not significant. Volume of distribution
of nicotine was similar for blacks and whites.

For cotinine, the total and nonrenal clearances were significantly lower
for blacks than for whites (Table 3).
Renal clearance of cotinine was higher in blacks than in whites. Half-life
of cotinine tended to be longer for blacks, while volume of distribution was
similar for both races. The fractional conversion of nicotine to cotinine
was similar for both.

Blacks and whites smoked similar numbers of cigarettes per day, but
blacks had higher baseline serum cotinine levels compared with whites (P=.06; Table 4).
The serum cotinine concentration per cigarette was 50% higher in blacks than
in whites (P=.003). The estimated daily intake of
nicotine from cigarette smoke was slightly greater, and the nicotine intake
per cigarette was 30% greater in blacks than in whites (P=.02, log transformed data).

No sex differences were found for nicotine or cotinine clearance or
fractional conversion of nicotine to cotinine. The volume of distribution
(VSS, measured in liters per kilogram) was greater respectively for men than
for women, ie, VSS=0.83±0.17 vs 0.72±0.10 (P<.001), and the half-life (t1/2, measured in minutes)
of cotinine was also greater in men than in women, ie, t1/2=1071±229
vs 943±315 (P=.04).

COMMENT

Our study confirms the observation made in previous studies that the
serum cotinine concentration per cigarette smoked is significantly higher
in black smokers than in white smokers.10,25
Our pharmacokinetic analysis indicates that this difference is attributable
to 2 factors. First, the clearance of cotinine is slower, leading to higher
levels of cotinine for a given level of nicotine intake in blacks compared
with whites. This difference is not attributable to systematic differences
in renal function by race, since all participants had normal serum creatinine
levels and blood pressure, and renal clearance is a minor pathway of nicotine
or cotinine clearance. Second, the intake of nicotine per cigarette tended
to be higher in blacks than in whites. Since intake of nicotine is highly
correlated to exposure to tar and oxidant gases, the latter observation may
help explain the higher smoking-related risks of lung cancer and reproductive
disorders in blacks compared with whites. However, the lower rate of COPD
among blacks is an unexplained paradox that may be related to other biological
or environmental factors.2

Pharmacokinetic analysis indicates that the nonrenal or metabolic clearance
of cotinine is significantly lower, and the metabolic clearance of nicotine
tends to be lower in blacks than in whites. One possible explanation for these
differences is that there may be a racial genetic difference in cotinine metabolism.
Blacks have been shown to metabolize some drugs at different rates than whites.26 Cotinine and nicotine appear to be metabolized primarily
by the enzyme CYP2A627,28 with
a lesser percentage conjugated via glucuronidation.29
No prior studies have looked at racial differences in drug metabolism via
CYP2A6 or glucuronidation. Our study represents the first reported racial
difference in the activity of one or both of these drug metabolizing enzymes.

Racial differences in drug-metabolizing activity could also be attributable
to environmental factors. One possible environmental explanation is that smoking
mentholated cigarettes influences cotinine metabolism. Nearly all of the blacks
and few of the whites in our study smoked mentholated cigarettes, which reflects
national racial patterns of smoking behavior.9,30
No data are available on the effects of menthol on drug metabolism, so further
research is needed to address this possibility.

The reasons why blacks take in more nicotine and more cigarette smoke
per cigarette are unclear. The most obvious possibility is that menthol via
its cooling action facilitates deep inhalation. However, studies measuring
puffing behavior and puff volumes after persons have smoked individual mentholated
vs nonmentholated cigarettes have not supported this explanation.31 Persons smoking mentholated cigarettes take fewer
puffs with lower average total volume of smoke, but with an increased carbon
monoxide boost compared with persons smoking regular cigarettes.31,32
Whether these observations are related to racial differences in nicotine intake
and cancer rates is not known.

Restriction of access to cigarettes has been shown to increase the intake
of nicotine per cigarette, believed to be a compensatory response to maintain
desired levels of nicotine in the body.33 It
is plausible to consider that economic constraints on purchasing cigarettes
among blacks lead to greater smoking of each cigarette, thereby increasing
intake of nicotine per cigarette compared with whites. However, cost of cigarettes
has not been found to be an important factor in motivating adults of any ethnic
group to quit smoking,34 and the proportion
of heavy smokers (≥25 cigarettes per day) in the United States is highest
among those with less than a high school education.7

Systematic differences in reporting number of cigarettes per day could
theoretically explain the difference in estimated nicotine intake per cigarette
by race. If blacks systematically underreported the number of cigarettes smoked
per day and whites were always accurate, we could be overestimating the nicotine
intake per cigarette by blacks. However, based on analyses from the National
Health Interview Surveys in 1970 and 1980, no evidence was found of a systematic
racial bias in self-reporting of cigarettes.35

The question of racial differences in self-reported use of cigarettes
compared with biochemical measures has been addressed in several studies.
Defining underreporting as a serum cotinine level of more than 142 nmol/L
(25 ng/mL) per cigarette smoked, we found that among Mexican Americans up
to 20% of men and 25% of women reporting 1 to 9 cigarettes per day were underreporters.36 Subsequently, using our definition of underreporting,
another study found that 75% of 95 black women smoking fewer than 20 cigarettes
per day were classified as underreporters.11
However, a recent study using carbon monoxide measures found no reporting
artifacts comparing white, African American, and Hispanic adolescents.37 Finally, 66 blacks and 97 whites were observed smoking
1 cigarette, had their cigarette butts counted for 1 week, and completed 2
self-reported measures of cigarette use 2 weeks apart, and the investigators
concluded that there was no evidence that underreporting differed by race.38 In that study blacks also had higher mean serum cotinine
levels and reported smoking fewer cigarettes per day.25

In summary, our study demonstrates, we believe for the first time, that
black smokers and white smokers differ in their metabolism of cotinine, as
well as in the intake of nicotine per cigarette. These differences have implications
in interpreting biomarkers of tobacco smoke exposure and possibly in explaining
differences in smoking-related disease risks in blacks and whites. Further
research in the possible role of mentholated cigarettes, differential effects
of tobacco use on disease rates, and comparison among other ethnic and racial
groups is needed.

US Department of Health and Human Services. Reducing the Health Consequences of Smoking: A Report of the
Surgeon General . Rockville, Md: US Dept of Health and Human Services, Public Health
Service, Centers for Disease Control, Center for Chronic Disease Prevention
and Health Promotion, Office on Smoking and Health; 1989.

Desenclos JCA, Hahn RA.Centers for Disease Control and Prevention. Years of potential life lost before age 65, by race Hispanic origin,
and sex—United States, 1986-1988. Mor Mortal Wkly Rep CDC Surveill Summ.1992;41(SS-6):13-23.